HIV vaccines comprised of multiple viral antigens are likely to be more effective in providing immunity that is sufficiently broad to recognize and control HIV upon exposure. Broad, sustained cellular and humoral immunity has been difficult to elicit with existing approaches, and both novel immunogens and more effective antigen presentation strategies are needed. Inactivated virus vaccines are attractive in principle, because they include all of the viral antigens in a native conformation and stoichiometry, and virus-like particles (VLPs) can elicit cellular immunity via cross-presentation. E2DISP, an engineered protein domain from Geobacillus stearothermophilus pyruvate dehydrogenase subunit E2, can be used to express N-terminal fusions of heterologous peptides or proteins. E2DISP self-assembles into 24 nm VLPs and can display up to 60 copies of a foreign antigen on the surface of the particle, and the E2DISP VLPs can be taken up by both the MHC Class I and Class II pathways to elicit both B cell and T cell immunity. Further, multiple foreign antigens can be displayed on the surface of a single particle, varying antigen copy number and particle composition. We have expressed most of the genome of HIV as eighteen fusion proteins with E2. In mice, E2Gag-p17 fusion protein particles are highly immunogenic and elicit very effective CD4 T helper responses, accelerating antibody responses in chimeric C57BL/6 x BALB/c F1 mice and CD8+ CTL in HLA-A2 transgenic mice. We propose to utilize the E2DISP antigen display system to explore the mechanisms, limitations, and applications for the expression and presentation of multiple HIV antigens and peptides as part of a prime-boost vaccine regimen. There are four aims: [unreadable] (1) Design novel HIV-E2 Envelope (Env) fusion proteins with strong and conserved neutralization determinants and determine their effectiveness in eliciting neutralizing antibodies (NAbs) in rabbits. [unreadable] (2) Explore immunization strategies to elicit long lasting Th1 CD4, CTL memory responses while preserving sustained antibody responses. [unreadable] (3) Explore optimal presentation and co-administration of E2DISP VLPs with C3d-based adjuvants in C57LI6/BALB/c mice. [unreadable] (4) Optimize the use of pure and hybrid E2-HIV VLPs in prime-boost regimens with systems effective in boosting cellular immunity and NAb responses in mice. [unreadable] We hypothesize that this approach will inform the development and optimal use of these novel immunogens for testing in nonhuman primates and humans to achieve long-lasting T- and B-cell responses in vivo. [unreadable] [unreadable] [unreadable]